Under the auspices of this small business innovation research (SBIR) grant, AuraSense LLC will develop a novel nanotechnology, "Nano-flares", for the detection of low abundance microRNA (miR) species in living cells in real-time. MicroRNAs are short (~22 nucleotides in length) non-protein coding RNA sequences that play a key role in regulating diverse cellular processes that occur, for instance, during development or during malignant transformation. Unique miRs are being evaluated as novel biomarkers for myriad diseases, and explored as novel therapeutic targets. No existing commercial technology is capable of detecting intracellular RNA target sequences, including miRs, in real-time in living cells. Nano-flares technology provides new opportunities for the detection of intracellular miR targets and will identify how these unique RNAs can be more effectively detected, improving our knowledge of miR function, and, ultimately targeted to improve human health. Nano-flare technology is based upon the unique conjugate properties of DNA oligonucleotides which can be densely loaded on the surface of gold nanoparticles (DNA-Au NPs). Importantly, DNA-Au NPs are universally taken up by cells and can be tailored to target specific intracellular RNA target sequences. Upon encountering a target RNA sequence, Nano-flares turn on a fluorescent signal which can be detected in single cells using fluorescent microscopy, or averaged over millions of cells using fluorescence activated cell sorting (FACS). In contrast to methods which employ the polymerase chain reaction (PCR), cells interrogated using the Nano-flares technology are alive and, thus, available for downstream applications. We propose using the Nano-flare technology to detect and quantify miR and messenger RNA (mRNA) sequences in live prostate cancer cells. Importantly, we will develop a cell model where specific miR and mRNA targets can be manipulated with small molecules in order to evaluate the ability of the Nano-flare system to detect and quantify relative changes in high and low abundance target miR. Results obtained with the Nano-flare system will be directly compared to those obtained using PCR techniques, the current commercial benchmark for detection of these targets. Project success will result in a robust platform for the detection of intracellular miR targets using FACS and confocal fluorescent microscopy. By using qPCR to confirm the Nano-flare results, this project will also set new analytical benchmarks for the real-time detection of miR target sequences inside of live cells, and define what 'low abundance'miR represents in single cells and in ensemble measurements. Finally, completion of this study will bring this enabling technology closer to commercialization and increase the potential for researchers and clinicians, ultimately improving patient health. PUBLIC HEALTH RELEVANCE: Despite the realized and increasing importance of microRNAs in human disease processes, no current methodology exists to detect changes in microRNA levels in live human cells in real-time. Nano-flare technology being developed by AuraSense, LLC will provide this capability, and represents a novel technology that is designed to provide PCR-like phenotypic characterization of intracellular microRNA targets, significantly, in living cells. Successful project completion will provide a unique technological tool for the study of microRNAs to the broader research community, and, ultimately, the clinical community to improve human health.